Chimeric antigen receptor-modified NK-92 cells targeting EGFR super-family receptors

ABSTRACT

Provided are genetically modified NK cells expressing a chimeric antigen receptor targeting an EGFR superfamily receptor. The CAR can comprise an intracellular domain of FcεRIγ and further recombinant proteins expressed by the genetically modified NK cells are CD16, autocrine growth stimulating cytokines, and optionally one of IL-12, a TGF-beta trap, or a homing receptor. Also described are methods for treating a patient having or suspected of having a disease that is treatable with NK-92 cells, such as cancer, comprising administering to the patient the genetically modified NK cells.

SEQUENCE LISTING

The content of the ASCII text file of the sequence listing namedSeq_listing_20200127_b_ST25, which is 61 kb in size was created on Jan.27, 2020 and electronically submitted via EFS-Web along with theapplication is incorporated by reference in its entirety.

FIELD OF THE INVENTION

The field of the invention is genetically modified immune competentcells that express a chimeric antigen receptor (CAR), and particularlymodified NK-92 cells expressing a CAR with an Fc epsilon receptor gamma(FcεRIγ) signaling domain targeting an EGFR superfamily receptor.

BACKGROUND OF THE INVENTION

Natural killer (NK) cells are cytotoxic lymphocytes that constitute asignificant component of the innate immune system. In most cases, NKcells represent about 10-15% of circulating lymphocytes, and bind andkill targeted cells, including virus-infected cells and many malignantcells. NK cell killing is non-specific with regard to particularantigens and can occur without prior immune sensitization. Killing oftargeted cells is typically mediated by cytolytic proteins, includingperforin, granzymes, and granulysin.

Autologous NK cells have been used as therapeutic entities. To that end,NK cells are isolated from the peripheral blood lymphocyte fraction ofwhole blood, expanded in cell culture to obtain sufficient numbers ofcells, and then re-infused into a subject. Autologous NK cells haveshown in at least some cases moderate effectiveness in both ex vivotherapy and in vivo treatment. However, isolation and growth ofautologous NK cell is time and cost intensive. Moreover, autologous NKcell therapy is further limited by the fact that not all NK cells arecytolytic.

At least some of these difficulties can be overcome by use of NK-92cells, which are a cytolytic cancer cell line which was discovered inthe blood of a subject suffering from a non-Hodgkins lymphoma and thenimmortalized in vitro (Gong et al., Leukemia 8:652-658 (1994)). WhileNK-92 cells are NK cell derivatives, NK-92 cells lack the major ofinhibitory receptors that are otherwise displayed by normal NK cells,and retain the majority of the activating receptors. NK-92 cells do not,however, attack normal cells nor do they elicit an unacceptable immunerejection response in humans. Due to these desirable characteristics,NK-92 cells were characterized in detail and explored as therapeuticagent in the treatment of certain cancers as is described, for example,in WO 1998/049268 or US 2002/068044.

Phenotypic changes distinguishing a tumor cell from normal cells derivedfrom the same tissue are often associated with one or more changes inthe expression of specific gene products, including the loss of normalcell surface components or the gain of others (i.e., antigens notdetectable in corresponding normal, non-cancerous tissue). The antigenswhich are expressed in neoplastic or tumor cells, but not in normalcells, or which are expressed in neoplastic cells at levelssubstantially above those found in normal cells, have been termed“tumor-specific antigens” or “tumor-associated antigens.” Suchtumor-specific antigens may serve as markers for tumor phenotype.Tumor-specific antigens include cancer/testis-specific antigen (e.g.MAGE, BAGE, GAGE, PRAME and NY-ESO-1), melanocyte differentiationantigens (e.g. tyrosinase, Melan-A/MART, gp100, TRP-1 and TRP-2),mutated or aberrantly expressed antigens (e.g. MUM-1, CDK4,beta-catenin, gp100-in4, p15 and N-acetylglucos-aminyltransferase V),and antigens that are expressed at higher levels in tumors (e.g., CD19and CD20).

Tumor-specific antigens have been used as targets for cancerimmunotherapies. One such therapy utilizes chimeric antigen receptors(CARs) expressed on the surface of immune cells, including T cells andNK cells, to improve cytotoxicity against cancer cells. CARs comprise asingle-chain variable fragment (scFv) linked to at least oneintracellular signaling domain. The scFv recognizes and binds an antigenon the target cell (e.g., a cancer cell) and triggers effector cellactivation. The signaling domains contain immunoreceptor tyrosine-basedactivation domains (ITAMs) that are important for intracellularsignaling by the receptor.

The first generation of CARs used in T-cells contained one cytoplasmicsignaling domain. For example, one version of a first-generation CAR inT-cells included a signaling domain from the Fc epsilon receptor gamma(FcεRIγ) which contained one ITAM, while another version contained thesignaling domain from CD3ζ which contained three ITAMs. In vivo and invitro studies showed that the CD3ζ CAR T-cells were more efficient attumor eradication than FcεRIγ CAR T-cells (e.g., Haynes, et al. 2001, J.Immunology 166:182-187; Cartellieri, et al. 2010, J. Biomed and Biotech,Vol. 2010, Article ID 956304). Additional studies then revealed thatcertain costimulatory signals were required for full activation andproliferation of such recombinant T-cells, and second and thirdgeneration CARs combined multiple signaling domains in to a single CARto enhance efficacy of the recombinant CAR T-cells. Due to their lessdesirable philological effects in the tested T-cells, first generationCARs and the FcεRIγ signaling domains were largely discarded in favor ofthe new, more efficient CARs using CD3ζ in combination with one or moreadditional signaling domains (e.g., Hermanson and Kaufman 2015,Frontiers in Immunol., Vol. 6, Article 195).

More recently, selected CARs have also been expressed in NK cells. Forexample, CAR-modified NK-92 cells have used first generation CARs withonly a CD3ζ intracellular signaling domain. Several antigens have beentargeted by these first generation CAR-NK cells, including CD19 and CD20for B cell lymphoma, ErbB2 for breast, ovarian, and squamous cellcarcinoma, GD2 for neuroblastoma, and CD138 for multiple myeloma. Secondgeneration CAR-NK cells from the NK-92 line have also been created forseveral antigens, including EpCAM for multiple carcinomas HLA-A2 EBNA3complex for Epstein-Barr virus, CS1 for multiple myeloma, and ErbB2 forHER2 positive epithelial cancers. The most common intracellularcostimulatory domain used alongside CD3ζ in second generation NK-92 CARsis CD28. However, the potential effect of the CD28 domain is unclearsince NK cells do not naturally express CD28. Additional secondgeneration CARs have incorporated the 4-1BB intracellular signalingdomain along with CD3ζ to improve NK cell persistence. Others comparedfunctionality of different intracellular domains using an ErbB2 scFvfused with CD3ζ alone, CD28 and CD3ζ, or 4-1BB and CD3ζ tested againstbreast cancer cells. They found that both of the second generationconstructs improved killing compared to the first generation CARs andthe CD28 and CD3ζ had 65% target lysis, the 4-1BB and CD3ζ lysed 62%,and CD3ζ alone killed 51% of targets. 4-1BB and CD28 intracellulardomains were also compared in a recent study using anti-CD19 CARsexpressed on NK-92 cells for B cell malignances. Still others found thatCD3ζ/4-1BB constructs were less effective than CD3ζ/CD28 in cell killingand cytokine production, highlighting differential effects of CD28 and4-1BB costimulatory domains.

Third generation NK-92 CARs were constructed of an anti-CD5 scFv withCD3ζ, CD28, and 4-1BB intracellular signaling domains and demonstratedspecific and potent anti-tumor activity against a variety of T-cellleukemia and lymphoma cell lines and primary tumor cells. Such cellswere also able to inhibit disease progression in xenograft mouse modelsof T cell Acute lymphoblastic leukemia (ALL) cell lines as well asprimary tumor cells (Transl Res. 2017 September; 187: 32-43). In furtherexamples, WO 2016/201304 and WO 2018/076391 teach use of thirdgeneration CD3ζ CARs expressed in NK cells and NK-92 cells.

However, NK cells (and particularly NK-92 cells) are often difficult togenetically modify as evidenced by numerous failures to engineer NK-92cells to express an Fc receptor. Such difficulties are furthercompounded where NK-92 cells are transfected with multiple recombinantgenes or relatively large recombinant nucleic acid payload forheterologous expression. Additionally, NK-92 cells also exhibit asignificant lack of predictability with respect to recombinantexpression of exogenous proteins (e.g., CD16). On a functional level,while exhibiting in most cases targeted cytotoxicity, most if not allCAR NK-92 cells require a high effector to target cell ratio.

In addition, even where cytotoxic cells expressing a CAR are relativelyeffective in vitro, various suppressive or inhibitory factors associatedwith the tumor microenvironment in vivo may reduce or even abrogatecytotoxicity of such cells. Similarly, and despite the expression of aCAR, such modified cells may not always be effective in targeting thetumor microenvironment.

Therefore, even though numerous recombinant NK-92 cells are known in theart, all or almost all of them suffer from various difficulties.Consequently, there remains a need for CAR-expressing NK-92 cells thatexpress a high-activity CAR in significant quantities, that can bereadily cultivated in a simple and effective manner, and that have highcytotoxicity in a tumor microenvironment.

SUMMARY OF THE INVENTION

The inventors have discovered that natural killer (NK) cells, andparticularly NK-92 cells, may be genetically modified to express atarget-specific CAR and further recombinant proteins to increase CARmediated cell killing, ADCC, and cytotoxicity in and/or homing to atumor microenvironment. In addition, such recombinant cells alsoexpressed cytokines for autocrine growth stimulation that advantageouslyassists in clonal selection of modified cells.

In one aspect of the inventive subject matter, the inventors contemplatea genetically modified NK cell (and especially an NK-92 cell) thatexpresses (i) a membrane bound recombinant chimeric antigen receptor(CAR) that comprises in a single polypeptide chain an extracellularbinding domain, a hinge domain, a transmembrane domain, and a signalingdomain, wherein the extracellular binding domain specifically binds toan EGFR superfamily receptor; (ii) a recombinant CD16; (iii) anautocrine growth stimulating cytokine; and (iv) optionally one of IL-12,a TGF-beta trap, or a homing receptor.

In some embodiments, the extracellular binding domain comprises a scFv,and/or the signaling domain comprises a FcεRIγ signaling domain. Mosttypically, the EGFR superfamily receptor is HER-2 or EGFR. In furthercontemplated aspects, the recombinant CD16 is a CD16158V mutant, and/orthe autocrine growth stimulating cytokine is IL-2 or IL-15, which mayadditionally comprise an endoplasmic retention sequence. In stillfurther contemplated embodiments, the IL-12 is a single chain IL-12heterodimer, and the TGF-beta trap comprises a single chain dimer of theTGF-beta Receptor II ectodomain and is preferably secreted. Preferredhoming receptors include cell adhesion molecules, selectins, integrins,a C—C chemokine receptor, or a C—X—C chemokine receptor. For example,suitable homing receptor include CCR1, CCR2, CCR3, CCR4, CCR5, CCR6,CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6,CXCR7, CX3CR1, XCR1, CCXCKR, D6, DARC, and the receptor for CXCL14.

Viewed from a different perspective, genetically modified NK cells arecontemplated that include a nucleic acid that encodes a (i) membranebound recombinant chimeric antigen receptor (CAR) that comprises in asingle polypeptide chain an extracellular binding domain, a hingedomain, a transmembrane domain, and a signaling domain, wherein theextracellular binding domain specifically binds to a EGFR superfamilyreceptor; (ii) a recombinant CD16; (iii) an autocrine growth stimulatingcytokine; and (iv) optionally one of IL-12, a TGF-beta trap, or a homingreceptor. With regard to the NK cell and the expressed proteins, thesame considerations as noted above apply.

In another aspect of the inventive subject matter, the inventors alsocontemplate a method of treating cancer in a patient in need thereof inwhich the patient receives a therapeutically effective amount of thegenetically modified NK cells presented herein, thereby treating thecancer. Where desired, contemplated methods may also include a step ofadministering at least one additional therapeutic entity selected fromthe group consisting of a viral cancer vaccine, a bacterial cancervaccine, a yeast cancer vaccine, N-803, an antibody, a stem celltransplant, and a tumor targeted cytokine. For example, contemplatedcancers include lung cancer, a breast cancer, a thyroid cancer, anesophageal cancer, a gastric cancer, a gastroesophageal cancer, or ahead and neck cancer. Most typically, about 1×10⁸ to about 1×10¹¹ cellsper m² of body surface area of the patient are administered to thepatient. Therefore, the inventors also contemplate use of a geneticallymodified NK cell in the treatment of cancer.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments along with the accompanying drawingfigures in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of exemplary CAR constructs. AllCAR variants had an extracellular domain comprising an anti-EGFRsuperfamily receptor scFv region (e.g., αEGFR-scFv, αHER2-scFv), a hingeregion from CD8 (CD8 hinge), and a transmembrane domain from CD28 (CD28TM). The intracellular domains of the CD19CARs were varied as indicated.

FIG. 2 is a schematic representation of an exemplary tricistronicrecombinant nucleic acid encoding a HER2.CAR, followed by a P2Asequence, followed by a sequence encoding a high-affinity variant ofCD16. The CD16 sequence is followed by an IRES sequence, which isfollowed in turn by a sequence encoding erIL-2.

FIG. 3 is another schematic representation of an exemplary tricistronicrecombinant nucleic acid encoding a HER2.CAR, a high-affinity variant ofCD16, and erIL-2 used to generate recombinant NK cells.

FIG. 4 depicts exemplary FACS results for NK cells transfected with thetricistronic recombinant nucleic acid demonstrating expression of CD16and HER2.CAR from a polyclonal collection of cells.

FIG. 5 depicts exemplary results for CAR-mediated cytotoxicity of NKcells transfected with the tricistronic recombinant nucleic acid of FIG.3.

FIG. 6 depicts exemplary results for ADCC of NK cells transfected withthe tricistronic recombinant nucleic acid of FIG. 3.

FIG. 7 depicts exemplary FACS results for NK cells transfected with thetricistronic recombinant nucleic acid demonstrating expression of CD16and HER2.CAR from selected clonal cell lines.

FIG. 8 depicts exemplary results for natural cytotoxicity of NK cellsfrom selected clonal cell lines transfected with the tricistronicrecombinant nucleic acid of FIG. 3.

FIG. 9 depicts exemplary results for CAR-mediated cytotoxicity of NKcells from selected clonal cell lines transfected with the tricistronicrecombinant nucleic acid of FIG. 3.

FIG. 10 depicts further exemplary results for CAR-mediated cytotoxicityof NK cells from selected clonal cell lines transfected with thetricistronic recombinant nucleic acid of FIG. 3.

FIG. 11 depicts further exemplary results for ADCC of NK cells fromselected clonal cell lines transfected with the tricistronic recombinantnucleic acid of FIG. 3.

FIG. 12 depicts exemplary results for erIL-2 release from NK cells fromselected clonal cell lines transfected with the tricistronic recombinantnucleic acid of FIG. 3.

FIG. 13 depicts exemplary in vivo results for MDA-MB-453 tumor volumechanges using NK cells transfected with the tricistronic recombinantnucleic acid of FIG. 3.

FIG. 14 depicts exemplary in vivo results for BT-474 tumor volumechanges using NK cells transfected with the tricistronic recombinantnucleic acid of FIG. 3.

FIG. 15 is a schematic representation of an exemplary tricistronicrecombinant nucleic acid encoding a EGFR2.CAR, followed by a P2Asequence, followed by a sequence encoding a high-affinity variant ofCD16. The CD16 sequence is followed by an IRES sequence, which isfollowed in turn by a sequence encoding erIL-2.

FIG. 16 is another schematic representation of an exemplary tricistronicrecombinant nucleic acid encoding a EGFR2.CAR, a high-affinity variantof CD16, and erIL-2 used to generate recombinant NK cells.

FIG. 17 depicts exemplary FACS results for NK cells transfected with thetricistronic recombinant nucleic acid demonstrating expression of CD16and EGFR2.CAR from a polyclonal collection of cells.

FIG. 18 depicts exemplary results from a polyclonal collection of cellsfor natural cytotoxicity of NK cells transfected with the tricistronicrecombinant nucleic acid of FIG. 16.

FIG. 19 depicts exemplary results from a polyclonal collection of cellsfor CAR-mediated cytotoxicity of NK cells transfected with thetricistronic recombinant nucleic acid of FIG. 16.

FIG. 20 depicts exemplary results from a polyclonal collection of cellsfor ADCC of NK cells transfected with the tricistronic recombinantnucleic acid of FIG. 16.

FIG. 21 depicts exemplary results from selected cell lines for naturalcytotoxicity of NK cells transfected with the tricistronic recombinantnucleic acid of FIG. 16.

FIG. 22 depicts exemplary results from selected cell lines forCAR-mediated cytotoxicity of NK cells transfected with the tricistronicrecombinant nucleic acid of FIG. 16.

FIG. 23 depicts further exemplary results from selected cell lines forCAR-mediated cytotoxicity of NK cells transfected with the tricistronicrecombinant nucleic acid of FIG. 16

FIG. 24 depicts further exemplary results for ADCC of NK cells fromselected clonal cell lines transfected with the tricistronic recombinantnucleic acid of FIG. 16.

FIG. 25 depicts exemplary results for erIL-2 release from NK cells fromselected clonal cell lines transfected with the tricistronic recombinantnucleic acid of FIG. 16.

FIG. 26 is a schematic representation of an exemplary quadracistronicrecombinant nucleic acid encoding a TGF-beta trap, a CAR, ahigh-affinity variant of CD16, and erIL-2 used to generate recombinantNK cells.

FIG. 27 depicts exemplary results for expression of the TGF-beta trap inNK cells transfected with the quadracistronic recombinant nucleic acidof FIG. 26.

FIG. 28 is a schematic representation of an exemplary quadracistronicrecombinant nucleic acid encoding an IL-12 heterodimer, a CAR, ahigh-affinity variant of CD16, and erIL-2 used to generate recombinantNK cells.

FIG. 29 is a schematic representation of IL-12 heterodimers andrespective expression levels of the IL-12 heterodimers in NK cellstransfected with the quadracistronic recombinant nucleic acid of FIG.28.

FIG. 30 is a schematic representation of an exemplary quadracistronicrecombinant nucleic acid encoding CCR7, a CAR, a high-affinity variantof CD16, and erIL-2 used to generate recombinant NK cells.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have discovered that genetically modified NK cells canhave significant general toxicity, target specific CAR-mediatedcytotoxicity, and ADCC (antibody dependent cellular cytotoxicity), andthat such genetically modified cells can be grown under autocrine growthstimulation that will also confer selective effect towards successfullytransfected cells. In addition, contemplated cells may further expressfrom a recombinant nucleic acid secreted IL-12 and/or a TGF-beta trap toreduce or eliminate an immune-suppressive environment. Additionally, oralternatively, contemplated cells may express from a recombinant nucleicacid a homing receptor. Therefore, modified cells may be generated bytransfection with a tricistronic or quadracistronic nucleic acid.

It should be further appreciated that FcεRIγ-containing CARs have notbeen utilized in NK-92 cells, other NK cell lines, or endogenous NKcells as other signaling domains (e.g., CD3ζ) were deemed moreefficient, especially when combined with additional signaling domains(in second and third generation CARs). Notably, the inventors havediscovered that NK-92 cells expressing a first-generation CAR comprisingan intracellular domain from FcεRIγ, which has only one ITAM domain,have equal or higher cytotoxic activity against cancer cells expressingthe antigen recognized by the CAR than NK-92 cells expressing CARs witha CD3ζ signaling domain, which has three ITAM domains, even where theseITAM domains were combined with other signaling domains (i.e., second orthird generation CARs). The inventors also made the unexpected findingthat a CAR comprising an intracellular domain from FcεRIγ was expressedat higher levels on the surface of NK-92 cells than other CARs,especially those comprising the CD3ζ signaling domain. Cytotoxic effectscan be even further enhanced by expression and secretion of IL-12, whichreduces immune suppression in the tumor microenvironment (e.g., viaexocrine stimulation of NK cells, modulation of MDSC, etc.), and/or byexpression and secretion or presentation of a TGF-beta trap.Alternatively, or additionally, cytotoxic effects can also be enhancedby expression of one or more homing receptors to attract and/or retainNK cells in the tumor microenvironment.

Therefore, in some aspects of the inventive subject matter, agenetically modified NK-92 cell or NK cell line is engineered to expressa chimeric antigen receptor (CAR) on a cell surface, and particularly aCAR that specifically binds to an EGFR superfamily receptor such as EGFRor HER2. Most typically, the CAR comprises an intracellular domain fromthe Fc epsilon receptor gamma (FcεRIγ). However, in further contemplatedembodiments the CAR may also comprise a T cell receptor (TCR) CD3ζ zeta(CD3ζ) intracellular domain, alone or in combination with additionalcomponents as are known from 2^(nd) and 3^(rd) generation CAR constructs(e.g., CD28, CD134, CD137, and/or ICOS). As will be readily appreciated,the CAR may be transiently or stably expressed by the NK-92 cell from arecombinant DNA or RNA molecule. Exemplary CAR constructs suitable foruse herein are shown in FIG. 1.

Consequently, in one aspect of the inventive subject matter, an NK cell,an NK-92 cell or NK/NK-92 cell line expresses a chimeric antigenreceptor (CAR) on the surface of the NK-92 cell that comprises acytoplasmic domain of FcεRIγ (e.g., having amino acid sequence of SEQ IDNO:1). Alternatively, or additionally, the CAR may also comprise acytoplasmic domain of CD3ζ zeta (e.g., having amino acid sequence of SEQID NO:7, which may be encoded by a nucleic acid of SEQ ID NO:8 (codonoptimized)). In another aspect, an NK or NK-92 cell line is contemplatedthat is transformed with a nucleic acid encoding a chimeric antigenreceptor (CAR). For example, preferred nucleic acids encode acytoplasmic domain of FcεRIγ (e.g., comprising or consisting of SEQ IDNO:2). As discussed in more detail below, the CAR may target a EGFRsuperfamily receptor such as EGFR or HER2.

In further contemplated embodiments, the NK, NK-92, or other NK cell ismodified to express an autocrine growth stimulating cytokine or variantthereof. For example, suitable cytokines may be transiently or stablyexpressed by the recombinant cell, and the cytokine may include anendoplasmic retention signal. Most typically (but not necessarily) aretention signal will reduce the amount of secreted cytokine and as suchmay act as an endocrine growth stimulus without producing systemiceffects otherwise encountered by the cytokine expression. Beneficially,the nucleic acid sequence encoding the autocrine growth stimulatingcytokine or variant thereof is located on the same recombinant nucleicacid, typically as part of a tri- or quadracistronic configuration.Consequently, recombinant cells transfected with the tri- orquadracistronic nucleic acid can be readily selected and propagated byvirtue of their independence from otherwise needed exogenous IL-2.

Additionally, it is generally preferred that the genetically modified NKcells will also express a recombinant CD16 or high-affinity variantthereof (e.g., CD16^(158V)) to impart to the cells target specific ADCC.Advantageously, such co-expression with the CAR is thought to furtherincrease cytotoxicity against a tumor cell. In this context, it shouldbe appreciated that unmodified NK cells typically do not express CD16and exhibit only cytotoxicity as a part of the innate immune system.

More recently it has been discovered that efficacy of immune therapy maybe reduced or even entirely abolished by various factors present in thetumor microenvironment. For example, immune suppressive factors includeamong other players certain cytokines (e.g., TGF-beta) and varioussuppressive cells (e.g., MDSC). Consequently, genetically modified NKcells may further express one or more recombinant proteins to counteractthe immune suppressive factors. For example, and as is described in moredetail below, the genetically modified NK cells may express a TGF-betatrap to reduce TGF-beta mediated effects in the tumor microenvironmentand/or may express IL-12 to suppress MDSCs. Additionally, oralternatively, the genetically modified NK cells may also express one ormore homing receptors to the tumor microenvironment (or other desiredtissue) to so increase the number of therapeutic cells in the tumormicroenvironment and thus enhance the therapeutic effect.

In another aspect of the inventive subject matter, the inventors alsocontemplate a method of treating cancer in a patient in need thereofthat includes a step of administering to the patient a therapeuticallyeffective amount of modified NK/NK-92 cells or an NK/NK-92 cell lineengineered to express a chimeric antigen receptor (CAR) as describedherein. Viewed form a different perspective, the inventors alsocontemplate a modified NK/NK-92 cell or a NK/NK-92 cell line thatexpresses a chimeric antigen receptor (CAR), preferably comprising acytoplasmic domain of FcεRIγ, for use in treating a tumor in a subject.In some embodiments, the use comprises administering to the subjecteffective amounts of modified cells or the cell line described herein totreat the tumor. In yet other embodiments, an in vitro method forkilling tumor cells is contemplated and may include a step of contactinga tumor cell with a modified NK-92 cell or NK-92 cell line describedherein. In some embodiments, the modified NK-92 cell or NK-92 cell lineexpresses a CAR that binds to an antigen on the tumor cell. In someembodiments, the CAR preferably comprises an intracellular domain fromthe Fc epsilon receptor gamma (FcεRIγ). Alternatively, or additionally,the CAR comprises a T cell receptor (TCR) CD3ζ zeta (CD3ζ) intracellulardomain.

With respect to suitable NK cells, it should be noted that all NK cellsare deemed suitable for use herein and therefore include primary NKcells (preserved, expanded, and/or fresh cells), secondary NK cells thathave been immortalized, autologous or heterologous NK cells (banked,preserved, fresh, etc.), and modified NK cells as described in moredetail below. In some embodiments, it is preferred that the NK cells areNK-92 cells. The NK-92 cell line is a unique cell line that wasdiscovered to proliferate in the presence of interleukin 2 (IL-2) (seee.g., Gong et al., Leukemia 8:652-658 (1994)). NK-92 cells are cancerousNK cells with broad anti-tumor cytotoxicity and predictable yield afterexpansion in suitable culture media. Advantageously, NK-92 cells havehigh cytolytic activity against a variety of cancers.

The original NK-92 cell line expressed the CD56^(bright), CD2, CD7,CD11a, CD45, and CD54 surface markers and did not display the CD1, CD3,CD4, CD5, CD8, CD10, CD14, CD16, CD19, CD20, CD23, and CD34 markers.Growth of such NK-92 cells in culture is dependent upon the presence ofinterleukin 2 (e.g., rIL-2), with a dose as low as 1 IU/mL beingsufficient to maintain proliferation. IL-7 and IL-12 do not supportlong-term growth, nor have various other cytokines tested, includingIL-1α, IL-6, tumor necrosis factor α, interferon α, and interferon γ.Compared to primary NK cells, NK-92 typically have a high cytotoxicityeven at relatively low effector:target (E:T) ratios, e.g. 1:1.Representative NK-92 cells are deposited with the American Type CultureCollection (ATCC), designation CRL-2407.

Therefore, suitable NK cells may have one or more modified KIR that aremutated such as to reduce or abolish interaction with MEW class Imolecules. Of course, it should be noted that one or more KIRs may alsobe deleted or expression may be suppressed (e.g., via miRNA, siRNA,etc.). Most typically, more than one KIR will be mutated, deleted, orsilenced, and especially contemplated KIR include those with two orthree domains, with short or long cytoplasmic tail. Viewed from adifferent perspective, modified, silenced, or deleted KIRs will includeKIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1,KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, KIR3DL3, andKIR3DS1. Such modified cells may be prepared using protocols well knownin the art. Alternatively, such cells may also be commercially obtainedfrom NantKwest (see URL www.nantkwest.com) as aNK cells (activatednatural killer cells). Such cells may then be additionally geneticallymodified to a CAR as further described in more detail below.

In another aspect of the inventive subject matter, the geneticallyengineered NK cell may also be an NK-92 derivative that is modified toexpress the high-affinity Fcγ receptor (CD16). Sequences forhigh-affinity variants of the Fcγ receptor are well known in the art(see e.g., Blood 2009 113:3716-3725; or SEQ ID NO:11 (with V at aminoacid position 158) and SEQ ID NO:12 (with codon for V at amino acidposition 158)), and all manners of generating and expression are deemedsuitable for use herein. Expression of such receptor is believed toallow specific targeting of tumor cells using antibodies that arespecific to a patient's tumor cells (e.g., neoepitopes), a particulartumor type (e.g., her2neu, PSA, PSMA, etc.), or that are associated withcancer (e.g., CEA-CAM). Advantageously, such antibodies are commerciallyavailable and can be used in conjunction with the cells (e.g., bound tothe Fcγ receptor). Alternatively, such cells may also be commerciallyobtained from NantKwest as haNK cells. Such cells may then beadditionally genetically modified to a CAR as further described in moredetail below.

Therefore, NK cells suitable for use herein include NK-92 cells (whichmay be transfected with a tricistronic or quadracistronic constructencoding a CAR, a CD16 or variant thereof, and a cytokine or variantthereof, and optionally one of IL-12, a TGF-beta trap, and a homingreceptor), a genetically modified NK cell or NK-92 cell that expresses aCD16 or variant thereof or a cytokine or variant thereof (which may betransfected with a nucleic acid encoding a CAR and a CD16 or variantthereof or a cytokine or variant thereof), and a genetically modified NKcell or NK-92 cell that expresses a CD16 or variant thereof and acytokine or variant thereof (which may be transfected with a nucleicacid encoding a CAR). As noted before, any of the NK cells contemplatedherein may express one of IL-12, a TGF-beta trap, and a homing receptorfrom the same or a different recombinant nucleic acid.

Genetic modification of the NK cells contemplated herein can beperformed in numerous manners, and all known manners are deemed suitablefor use hereon. Moreover, it should be recognized that NK cells can betransfected with DNA or RNA, and the particular choice of transfectionwill at least in part depend on the type of desired recombinant cell andtransfection efficiency. For example, where it is desired that NK cellsare stably transfected, linearized DNA may be introduced into the cellsfor integration into the genome. On the other hand, where transienttransfection is desired, circular DNA or linear RNA (e.g., mRNA withpolyA⁺ tail) may be used.

Similarly, it should be appreciated that the manner of transfection willat least in part depend on the type of nucleic acid employed. Therefore,viral transfection, chemical transfection, mechanical transfectionmethods are all deemed suitable for use herein. For example, in oneembodiment, the vectors described herein are transient expressionvectors. Exogenous transgenes introduced using such vectors are notintegrated in the nuclear genome of the cell; therefore, in the absenceof vector replication, the foreign transgenes will be degraded ordiluted over time.

In another embodiment, vectors will preferably allow for stabletransfection of cells. In one embodiment, the vector allowsincorporation of the transgene(s) into the genome of the cell.Preferably, such vectors have a positive selection marker and suitablepositive selection markers include any genes that allow the cell to growunder conditions that would kill a cell not expressing the gene.Non-limiting examples include antibiotic resistance, e.g. geneticin (Neogene from Tn5). Alternatively, or additionally, the vector is a plasmidvector. In one embodiment, the vector is a viral vector. As would beunderstood by one of skill in the art, any suitable vector can be used,and suitable vectors are well-known in the art.

In still other embodiments, the cells are transfected with mRNA encodingthe protein of interest (e.g., the CAR). Transfection of mRNA results intransient expression of the protein or proteins. In one embodiment,transfection of mRNA into NK-92 cells is performed immediately prior toadministration of the cells. In one embodiment, “immediately prior” toadministration of the cells refers to between about 15 minutes and about48 hours prior to administration. Preferably, mRNA transfection isperformed about 5 hours to about 24 hours prior to administration. In atleast some embodiments as described in more detail below, NK celltransfection with mRNA resulted in unexpectedly consistent and strongexpression of the CAR at a high faction of transfected cells. Moreover,such transfected cells also exhibited a high specific cytotoxicity atcomparably low effector to target cell ratios.

With respect to contemplated CARs it is noted that the NK/NK-92 cellswill be genetically modified to express the CAR as a membrane boundprotein exposing a portion of the CAR on the cell surface whilemaintaining the signaling domain in the intracellular space. Mosttypically, the CAR will include at least the following elements (inorder): an extracellular binding domain, a hinge domain, a transmembranedomain, and a signaling domain (preferably, but not necessarily a FcεRIγdomain).

In preferred embodiments, the cytoplasmic domain of the CAR comprises orconsists of a signaling domain of FcεRIγ. For example, the FcεRIγsignaling domain comprises or consists of or consists essentially of theamino acid sequence of SEQ ID NO:1. In some embodiments, the FcεRIγcytoplasmic domain is the sole signaling domain. However, it should beappreciated that additional elements may also be included, such as othersignaling domains (e.g., CD28 signaling domain, CD3ζ signaling domain,4-1BB signaling domain, etc.). These additional signaling domains may bepositioned downstream of the FcεRIγ cytoplasmic domain and/or upstreamof the FcεRIγ cytoplasmic domain.

In some embodiments, the FcεRIγ signaling domain comprises or consistsof or consists essentially of an amino acid sequence having at leastabout 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology to theamino acid sequence of SEQ ID NO:1.

As noted above, in some embodiments, the cytoplasmic domain of the CARcomprises a signaling domain of CD3 zeta (CD3ζ). In one embodiment, thecytoplasmic domain of the CAR consists of a signaling domain of CD3zeta. In one embodiment, the CD3 zeta signaling domain comprises orconsists of or consists essentially of the amino acid sequence of SEQ IDNO:7. In some embodiments, the CD3 zeta signaling domain comprises orconsists of or consists essentially of an amino acid sequence having atleast about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology tothe amino acid sequence of SEQ ID NO:7.

The CAR may comprise any suitable transmembrane domain. In one aspect,the CAR comprises a transmembrane domain of CD28. In one embodiment, theCD28 transmembrane domain comprises or consists of or consistsessentially of the amino acid sequence of SEQ ID NO:4. In oneembodiment, the CD28 transmembrane domain comprises or consists of orconsists essentially of an amino acid sequence having at least about85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology to the amino acidsequence of SEQ ID NO:4. In one embodiment, the transmembrane domain isselected from a CD28 transmembrane domain, 4-1BB transmembrane domain,or FcεRIγ transmembrane domain.

The CAR may comprise any suitable hinge region. In one aspect, the CARcomprises a hinge region of CD8. In one embodiment, the CD8 hinge regioncomprises or consists of or consists essentially of the amino acidsequence of SEQ ID NO:3. In one embodiment, the CD8 hinge regioncomprises or consists of or consists essentially of an amino acidsequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence homology to the amino acid sequence of SEQ ID NO:3.

Most typically, but not necessarily, the extracellular binding domain ofthe CAR will be a scFv or other natural or synthetic binding portionthat specifically binds an antigen of interest such as an EGFRsuperfamily receptor (and particularly EGFR or HER2). Especiallysuitable binding portions include small antibody fragments with single,dual, or multiple target specificities, beta barrel domain binders, pagedisplay fusion proteins, etc. However, in other embodiments, suitableextracellular binding domains will specifically bind to a tumor-specificantigen, a tumor associated antigen, or a patient- and tumor-specificantigen. For example, contemplated antigens include CD19, CD20, GD2,HER-2, CD30, EGFR, FAP, CD33, CD123, PD-L1, IGF1R, CSPG4, or B7-H4.Further tumor-specific antigens are described, by way of non-limitingexample, in US2013/0189268; WO 1999024566 A1; U.S. Pat. No. 7,098,008;and WO 2000020460, each of which is incorporated herein by reference inits entirety.

Therefore, contemplated CARs will generally have a structure of anextracellular binding domain that is (directly) coupled to a hingedomain, which is (directly) coupled to a transmembrane domain, which is(directly) coupled to a (e.g., FcεRIγ) signaling domain. In stillfurther contemplated aspects, contemplated CARs may also include one ormore signaling domains in addition to or replacing the FcεRIγ signalingdomain, and especially contemplated signaling domains include CD3ζsignaling domains, 4-1BB signaling domains, and CD28 signaling domains.For example, contemplated CAR polypeptides may therefore include any oneof the binding domains having SEQ ID NO:41 (EGFR scFv—amino acidsequence; encoded by a nucleic having SEQ ID NO:40, or a sequence havingat least 90%, or at least 95% or at least 96%, or at least 97%, or atleast 98%, or at least 99% identity to SEQ ID NO:40) and SEQ ID NO:43(HER2/neu scFv—amino acid sequence; encoded by a nucleic having SEQ IDNO:42, or a sequence having at least 90%, or at least 95% or at least96%, or at least 97%, or at least 98%, or at least 99% identity to SEQID NO:42) that is coupled to a hinge domain (e.g., CD8 hinge as in SEQID NO:3), which is in turn coupled to a transmembrane domain (e.g., CD28TM as in SEQ ID NO:4), which is coupled to a signaling domain (e.g.,FcεRIγ signaling domain as in SEQ ID NO:1, CD28 signaling domain as inSEQ ID NO:5, 4-1BB signaling domain as in SEQ ID NO:6, and/or CD3ζsignaling domain as in SEQ ID NO:7)

With respect to the construction of contemplated CARs it should berecognized that CARs can be engineered in numerous manners as described,for example, in WO 2014/039523; US 2014/0242701; US 2014/0274909; US2013/0280285 and WO 2014/099671, each of which is incorporated herein byreference in its entirety.

Viewed from a different perspective, contemplated CARs target an antigenassociated with a specific cancer type, and more particularly cancersthat overexpress EGFR and/or HER2 such as lung cancer (e.g., small cell)and breast cancer (e.g., TBNC), thyroid cancer, esophageal cancer,gastric cancer, gastroesophageal cancer, head and neck cancer, etc.

In still further contemplated aspects, NK cells may be furthergenetically modified to express one or more cytokines, and especially anautocrine growth stimulating cytokine to so provide a selection markerwhere the cytokine and the CAR are encoded on the same recombinantnucleic acid and/or to render the recombinant cells independent ofexogenous IL-2. Therefore, in some embodiments, NK-92 cells are modifiedto express at least one cytokine. In particular, the at least onecytokine is IL-2, IL-12, IL-15, IL-18, IL-21, or a variant thereof. Inpreferred embodiments, the cytokine is IL-2 or a variant thereof andespecially preferred variants include endoplasmic retention signals(e.g., human IL-2 polypeptide as in SEQ ID NO:9, optionally with ERretention signal as in SEQ ID NO:10). For example, the IL-2 gene iscloned and expressed with a signal sequence that directs the IL-2 to theendoplasmic reticulum. This permits expression of IL-2 at levelssufficient for autocrine activation, but without releasing IL-2extracellularly (e.g., Exp Hematol. 2005 February; 33(2):159-64.)Alternatively, expression of a cytokine (and especially IL-15) may alsobe such that the cytokine will be expressed in sufficient quantities toprovide an autocrine growth signal to the recombinant cells, but also toallow at least some of the expressed IL-15 to be released from the cell,which will so provide an immune stimulatory signal. For example, suchexpression may be achieved using a human IL-15 sequence that includesboth the signal peptide and an endoplasmic retention sequence. Anexemplary DNA and protein sequence for an endoplasmic retained IL-15 isshown in SEQ ID NO:38 and SEQ ID NO:39, respectively.

Where desired, contemplated cells may also express a suicide gene. Theterm “suicide gene” refers to a transgene that allows for the negativeselection of cells expressing the suicide gene. A suicide gene is usedas a safety system, allowing cells expressing the gene to be killed byintroduction of a selective agent. This is desirable in case therecombinant gene causes a mutation leading to uncontrolled cell growth,or the cells themselves are capable of such growth. A number of suicidegene systems have been identified, including the herpes simplex virusthymidine kinase (TK) gene, the cytosine deaminase gene, thevaricella-zoster virus thymidine kinase gene, the nitroreductase gene,the Escherichia coli gpt gene, and the E. coli Deo gene. Typically, thesuicide gene encodes for a protein that has no ill effect on the cellbut, in the presence of a specific compound, will kill the cell. Thus,the suicide gene is typically part of a system.

In one embodiment, the suicide gene is active in NK-92 cells. In oneembodiment, the suicide gene is the thymidine kinase (TK) gene. The TKgene may be a wild-type or mutant TK gene (e.g., tk30, tk75, sr39tk).Cells expressing the TK protein can be killed using ganciclovir. Inanother embodiment, the suicide gene is cytosine deaminase, which istoxic to cells in the presence of 5-fluorocytosine. Garcia-Sanchez etal. “Cytosine deaminase adenoviral vector and 5-fluorocytosineselectively reduce breast cancer cells 1 million-fold when theycontaminate hematopoietic cells: a potential purging method forautologous transplantation.” Blood. 1998 Jul. 15; 92(2):672-82. In afurther embodiment, the suicide gene is cytochrome P450, which is toxicin the presence of ifosfamide or cyclophosphamide. See, e.g. Touati etal. “A suicide gene therapy combining the improvement ofcyclophosphamide tumor cytotoxicity and the development of an anti-tumorimmune response.” Curr Gene Ther. 2014; 14(3):236-46. In yet anotherembodiment, the suicide gene is iCasp9. Di Stasi, (2011) “Inducibleapoptosis as a safety switch for adoptive cell therapy.” N Engl J Med365: 1673-1683. See also Morgan, “Live and Let Die: A New Suicide GeneTherapy Moves to the Clinic” Molecular Therapy (2012); 20: 11-13. iCasp9induces apoptosis in the presence of a small molecule, AP1903. AP1903 isbiologically inert small molecule, that has been shown in clinicalstudies to be well tolerated, and has been used in the context ofadoptive cell therapy.

Where the modified NK cells are further engineered to express IL-12, itis generally preferred that the IL-12 is expressed as a single chainheterodimer in which the p35 and p40 components are linked together by aflexible linker (in either orientation, p35-linker-p40 orp40-linker-p35). Moreover, it is generally preferred that theheterodimer will be secreted and as such may include a signal peptidefor protein export. Therefore, suitable IL-12 sequences as contemplatedherein may comprise a nucleic acid sequence with at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:26(p35 n.t. sequence), or SEQ ID NO:28 (p40 n.t. sequence). The IL-12contemplated herein may also comprise an amino acid sequence with atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identity to SEQ ID NO:27 (p35 a.a. sequence, isoform 1 precursor), orSEQ ID NO:29 (p40 a.a. sequence, precursor). Thus, the IL-12 singlechain p40 p35 sequence may comprise a polypeptide sequence with at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity toSEQ ID NO:31, or may comprise an polynucleotide sequence with at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity toSEQ ID NO:32. Most preferably, but not necessarily, the nucleic acidencoding the IL-12 single chain heterodimer will be part of apolycistronic nucleic acid sequence (e.g., present as a quadracistronicsequence with CAR, CD16, and erIL-2).

Where the modified NK cells are further engineered to express a TGF-betatrap, it is generally preferred that the TGF-beta trap is a single chaindimer of the extracellular domain of a TGFβRII molecule, and mostpreferably comprises a single chain dimer of the TGF-beta Receptor IIectodomain. Suitable TGF-beta traps are therefore encoded by apolynucleotide sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:34 (TGFBRIIextracellular domain), or SEQ ID NO:36 (TGF beta trap sequence). TheTGF-beta trap contemplated herein may also comprise an amino acidsequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identity to SEQ ID NO:35 (TGFBRII extracellular domain), orSEQ ID NO:37 (TGF-beta trap sequence). Other suitable TGF-beta trapsinclude those described in Mol. Canc. Ther. 2012, Vol 11(7), 1477-1487.Most preferably, but not necessarily, the nucleic acid encoding theTGF-beta trap will be part of a polycistronic nucleic acid sequence(e.g., present as a quadracistronic sequence with CAR, CD16, anderIL-2).

Where the modified NK cells are further engineered to express a homingreceptor it is noted that the term “homing receptor” refers to areceptor that activates a cellular pathway that results directly orindirectly in the cell migrating toward a target cell or tissue. Forexample, homing receptors expressed by leukocytes are used by leukocytesand lymphocytes to enter secondary lymphoid tissues via high endothelialvenules. Homing receptors can also be used by cells to migrate towardthe source of a chemical gradient, such as a chemokine gradient.Examples of homing receptors include G-protein coupled receptors such aschemokine receptors, including CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7,CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7,CX3CR1, XCR1, CCXCKR, D6, and DARC; cytokine receptors; cell adhesionmolecules such as selectins, including L-selectin (CD62L), integrinssuch as α4β7 integrin, LPAM-1, and LFA-1. Homing receptors generallybind to cognate ligands on the target tissues or cell. In someembodiments, homing receptors bind to addressins on the endothelium ofvenules, such as mucosal vascular addressing cell adhesion molecule 1(MAdCAM-1).

In some exemplary embodiments, the chemokines and homing receptorscontemplated herein may comprise a polypeptide sequence or apolynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:13 (CCR7 n.t.sequence), or SEQ ID NO:14 (CCL19 n.t. sequence), or SEQ ID NO:15 (CCL21n.t. sequence), or SEQ ID NO:16 (CXCR2 n.t. sequence), or SEQ ID NO:17(CXCR2 a.a. sequence), or SEQ ID NO:18 (CXCL14 n.t. sequence), or SEQ IDNO:19 (CXCL4 a.a. sequence), or SEQ ID NO:20 (CD62L n.t. sequence), orSEQ ID NO:21 (CD62L a.a. sequence), or SEQ ID NO:22 (IL-8 n.t.sequence), or SEQ ID NO:23 (IL-8 a.a. sequence), or SEQ ID NO:24 (CXCL1n.t. sequence), or SEQ ID NO:25 (CXCL1 a.a. sequence). Most preferably,but not necessarily, the nucleic acid encoding the homing receptor willbe part of a polycistronic nucleic acid sequence (e.g., present as aquadracistronic sequence with CAR, CD16, and erIL-2).

Of course, it should be noted that all of the recombinant proteins canbe expressed from individual recombinant sequences. However, it isgenerally preferred that where multiple recombinant sequences areexpressed (e.g., CAR, CD16, cytokine, TGF-beta trap), coding regions maybe arranged in a polycistronic unit with at least two or at least threeor at least four coding regions encoding the recombinant proteins.Therefore, transgenes can be engineered into an expression vector by anymechanism known to those of skill in the art. Where multiple transgenesare to be inserted into a cell, transgenes may be engineered into thesame expression vector or a different expression vector. In someembodiments, the cells are transfected with mRNA encoding the transgenicprotein to be expressed. In some embodiments, the cells are transfectedwith DNA encoding the transgenic protein to be expressed. Transgenes,mRNA and DNA can be introduced into the NK-92 cells using anytransfection method known in the art, including, by way of non-limitingexample, infection, viral vectors, electroporation, lipofection,nucleofection, or “gene-gun.”

In preferred embodiments, it should therefore be noted that thegenetically modified NK cell (especially where the cell expresses a CARand CD16 or variant thereof) will exhibit three distinct modes of cellkilling: General cytotoxicity which is mediated by activating receptors(e.g., an NKG2D receptor), ADCC which is mediated by antibodies bound toa target cell, and CAR mediated cytotoxicity. Where desired, therapeuticeffect in the tumor microenvironment may be further increased viaexpression and secretion of IL-12 (e.g., as a single chain heterodimer),via expression and presentation/secretion of a TGF-beta trap (e.g., as asingle chain dimer of the TGF-beta Receptor II ectodomain) or viaexpression of a homing receptor (e.g., CCR7). As will be readilyapparent, contemplated genetically modified cells can be used fortreatment of various diseases, and especially of various cancers andviral infections where a diseased cell presents a disease-specific ordisease-associated antigen. Consequently, the inventors contemplatemethods of treating patients with modified NK or NK-92 cells asdescribed herein. In one embodiment, the patient is suffering fromcancer (e.g., a tumor) and the modified NK-92 cell or cell lineexpresses a CAR specific for an antigen expressed on the surface of acell from the cancer or tumor. As noted above, in some embodiments, thecancer is lung cancer, a breast cancer, a thyroid cancer, an esophagealcancer, a gastric cancer, a gastroesophageal cancer, or a head and neckcancer.

Contemplated modified NK or NK-92 cells can be administered to anindividual by absolute numbers of cells. For example, the individual canbe administered from about 1000 cells/injection to up to about 10billion cells/injection, such as at about, at least about, or at mostabout, 1×10⁸, 1×10⁷, 5×10⁷, 1×10⁶, 5×10⁶, 1×10⁵, 5×10⁵, 1×10⁴, 5×10⁴,1×10³, 5×10³ (and so forth) modified NK-92 cells per injection, or anyranges between any two of the numbers, end points inclusive. In otherembodiments, modified NK-92 cells can be administered to an individualby relative numbers of cells, e.g., said individual can be administeredabout 1000 cells to up to about 10 billion cells per kilogram of theindividual, such as at about, at least about, or at most about, 1×10⁸,1×10⁷, 5×10⁷, 1×10⁶, 5×10⁶, 1×10⁵, 5×10⁵, 1×10⁴, 5×10⁴, 1×10³, 5×10³(and so forth) modified NK-92 cells per kilogram of the individual, orany ranges between any two of the numbers, end points inclusive. Inother embodiments, the total dose may be calculated by m² of bodysurface area, including about 1×10¹¹, 10¹⁰, 1×10⁹, 1×10⁸, 1×10⁷, per m²,or any ranges between any two of the numbers, end points inclusive. Theaverage person is about 1.6 to about 1.8 m². In a preferred embodiment,between about 1 billion and about 3 billion NK-92 cells are administeredto a patient.

Modified NK-92 cells, and optionally other anti-cancer agents can beadministered once to a patient with cancer or infected with a virus orcan be administered multiple times, e.g., once every 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours,or once every 1, 2, 3, 4, 5, 6 or 7 days, or once every 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more weeks during therapy, or any ranges between anytwo of the numbers, end points inclusive.

In one embodiment, where the modified NK cells express a suicide gene,the patient is administered an agent to trigger modified NK cell death.In one embodiment, the agent is administered at a time point afteradministration of the modified NK cells that is sufficient for the NKcells to kill target cells.

In one embodiment, the modified NK cells are irradiated prior toadministration to the patient. Irradiation of NK cells is described, forexample, in U.S. Pat. No. 8,034,332, which is incorporated herein byreference in its entirety. In one embodiment, modified NK cells thathave not been engineered to express a suicide gene are irradiated.

Furthermore, it should be appreciated that contemplated treatments willalso include administration of other immune therapeutic entities, andespecially preferred immune therapeutic entities include a viral cancervaccine (e.g., adenoviral vector encoding cancer specific antigens), abacterial cancer vaccine (e.g., non-pyrogenic E. coli expressing one ormore cancer specific antigens), a yeast cancer vaccine, N-803 (alsoknown as ALT-803, ALTOR Biosciences), an antibody (e.g., binding to atumor associated antigen or patient specific tumor neoantigen), a stemcell transplant (e.g., allogeneic or autologous), and a tumor targetedcytokine (e.g., NHS-IL12, IL-12 coupled to a tumor targeting antibody orfragment thereof).

After reading this description, it will become apparent to one skilledin the art how to implement the invention in various alternativeembodiments and alternative applications. However, not all embodimentsof the present invention are described herein. It will be understoodthat the embodiments presented here are presented by way of an exampleonly, and not limitation. As such, this detailed description of variousalternative embodiments should not be construed to limit the scope orbreadth of the present invention as set forth below.

Before aspects of the present inventive subject matter are disclosed anddescribed in more detail, it is to be understood that the aspectsdescribed below are not limited to specific compositions, methods ofpreparing such compositions, or uses thereof as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular aspects only and is notintended to be limiting.

EXAMPLES

The following examples are for illustrative purposes only and should notbe interpreted as limitations of the claimed invention. There are avariety of alternative techniques and procedures available to those ofskill in the art which would similarly permit one to successfullyperform the intended invention.

Example 1: HER2.CAR t-haNK Cells

Unless specified otherwise in the following examples, the inventors usedNK-92 cells for all transfections of the cells with recombinant nucleicacids. Furthermore, and also unless noted otherwise, all recombinantnucleic acids were linearized DNA constructs encoding a tri- orquadracistronic configuration.

To generate HER2.CAR t-haNK cells, a recombinant DNA molecule wasassembled as is schematically depicted in FIG. 2 where the tricistronicconfiguration included a sequence encoding a HER2.CAR followed by a P2Asequence, which was followed by a sequence encoding CD16 (orCD16^(158V)), and which in turn was followed by an IRES sequence elementupstream of a sequence encoding erIL-2. Unless otherwise noted, theHER2.CAR had a structure as exemplarily shown in FIG. 1 (however, withan Fc epsilon signaling sequence) and had a nucleic acid sequence of SEQID NO:30. Of course, it should be noted that other suitable sequencesfor the production of HER2.CAR may have a sequence identity of at least90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%,or at least 99% to SEQ ID NO:30. The primary transcript of thetricistronic nucleic acid then lead to the formation of HER2.CAR,CD16¹⁵⁸, and erIL-2 as the recombinant polypeptides. FIG. 3 exemplarilyand schematically depicts a linearized recombinant nucleic acid used fortransfection of the NK-92 cells.

All transfections generally followed standard protocol: NK-92 cells weregrown in X-Vivo10 medium (Lonza, Basel, Switzerland) supplemented with5% Human AB Serum (Valley Biomedical, Winchester, Va.) and 500 IU/mLIL-2 (Prospec, Rehovot, Israel). Cells were electroporated withtricistronic or quadracistronic DNA using the Neon™ electroporationdevice (Life Technologies, Carlsbad, Calif.), following themanufacturer's parameters for NK-92 cells (1250 V, 10 ms, 3 pulses) andusing 5 μg of DNA per 10⁶ cells in a volume of 100 μl. Electroporatedcells were transferred into medium (same as above) without exogenouslyadded IL-2.

Selection for recombinant cells and clones was based on continued cellculture in the absence of exogenously added IL-2 as all recombinantcells included a recombinant autocrine growth promoting cytokine (e.g.,IL-2, erIL-2, IL-15, or erIL-15).

The HER2.CAR and CD16 expression on the NK-92 cell surface wasdetermined by flow cytometry using biotinylated soluble HER2 protein andAPC-labeled streptavidin, and fluorescently labeled anti-CD16 antibody.Exemplary results for HER2.CAR and CD16 expression (polyclonal) areshown in FIG. 4 along with aNK (not expressing CD16) and haNK(expressing CD16) controls. As can be readily seen from FIG. 4, thepolyclonal cell cultures had a significant and strong expression forboth, HER2.CAR and CD16^(158V). In this context, it should be noted thatthe HER2.CAR was expressed at notably higher levels where the HER2.CARhad a FcεRIγ signaling portion as compared to other signaling portions(data not shown).

HER2.CAR t-haNK cells had strong and target specific CAR-mediatedcytotoxicity as can be seen from the results depicted in FIG. 5. Here,SKBR3 cells and SUP-B15^(HER−2+) cells were co-incubated with HER2.CARt-haNK cells at varying effector to target cell ratios (e.g., between0.075 and 10) in a flow-cytometry based in vitro cytotoxicity assay, andtarget specificity was compared to aNK cells as indicated. As can beseen, CAR-mediated cytotoxicity using a polyclonal HER2.CAR t-haNK cellpopulation was >60% for SKBR3cells and approaching 90% forSUP-B15^(HER−2+) cells.

In a similar manner, ADCC was tested using SUP-B15^(HER−2−/CD20+) cellsand rituximab as target specific antibody and Herceptin as controlantibody. Once again, the polyclonal HER2.CAR t-haNK cells had strongand target specific ADCC approaching 60% as can be seen from theexemplary results depicted in FIG. 6, with no substantial off-targettoxicity.

A number of individual clones from the HER2.CAR t-haNK cell populationwere then prepared following dilution propagation, and expressionanalysis was once more performed via FACS using the same procedure asdescribed above. aNK and haNK cells were used as controls. Once more, itwas observed that all individual clones had a significant and strongexpression of both HER2.CAR and CD16^(158V) as can be seen from theexemplary results of FIG. 7.

Individual HER2.CAR t-haNK cell clones were tested for naturalcytotoxicity using K562 cells and results were compared tonon-transfected aNK cells as is shown in FIG. 8. Here, specific lysis ateffector to target cell ratios as indicated was lower than control aNKlysis, however, only about 10-20% less. On the other hand, CAR-mediatedcytotoxicity was once more substantial and target restricted usingSUP-B15^(HER−2+) cells and exemplary results for specific HER2.CARt-haNK cell clones are shown in FIG. 9 over a wide range of effector totarget ratios. Likewise, where SKBR3 cells were used as target cells,notable CAR-mediated cytotoxicity was again observed for all of thetested HER2.CAR t-haNK cell clones as is exemplarily shown in FIG. 10.Selected HER2.CAR t-haNK cell clones were also tested for ADCC againstSUP-B15^(HER−2−/CD20+) cells using rituximab as target specific antibodyand Herceptin as control antibody. As can be taken from FIG. 11, allHER2.CAR t-haNK cell clones had once more significant, strong, andtarget specific ADCC against control.

While all HER2.CAR t-haNK polyclonal cultures and cell clones propagatedwell in the absence of exogenous IL-2, expression of er-IL2 was alsotested along with the quantity of erIL-2 released in the culture medium.Exemplary results are shown in FIG. 12 depicting a minor release oferIL-2 into the culture medium. Notably, the extracellular erIL-2release was significantly less than that of a closely related haNK003cell line as is shown in FIG. 12. Such reduced release may furtheradvantageously reduce potential systemic effects otherwise attributableto IL-2 (e.g., vascular permeability, systemic vascular leak, etc.).

To test in vivo efficacy of the HER2.CAR t-haNK cells, NSG mice(NOD.Cg-Prkdc^(scid) Il2rg^(tm1Wjl)/SzJ) were implanted with MDA-MB-453tumor cells subcutaneously, and tumor volume was measured. When tumorsreached an average ˜120 mm³, HER2.CAR t-haNK cells were administeredintratumorally or intravenously twice a week for the duration of thestudy. Following administration of HER2.CAR t-haNK cells, tumor volumewas sustainably reduced compared to vehicle control treatment as isdemonstrated by the exemplary results in FIG. 13. It should beappreciated that intratumoral administration required significantly lessHER2.CAR t-haNK cells for the same reduction in tumor volume thansystemic intravenous administration.

Similarly, in vivo efficacy of the HER2.CAR t-haNK cells was tested inNSG mice that were implanted with BT-474 tumor cells subcutaneously, andthe tumor volume was measured over time. When tumors reached an average˜120 mm³, HER2.CAR t-haNK cells were administered intratumorally orintravenously twice a week for the duration of the study. Followingadministration of HER2.CAR t-haNK cells, the increase in tumor volumewas substantially reduced over the time period tested as is shown by theexemplary results in FIG. 14. Once more, it should be appreciated thatintratumoral administration required significantly less HER2.CAR t-haNKcells for the same reduction in tumor growth than systemic intravenousadministration.

Example 2: EGFR.CAR t-haNK Cells

To generate EGFR.CAR t-haNK cells, a recombinant DNA molecule wasassembled as is schematically depicted in FIG. 15 where the tricistronicconfiguration included a sequence encoding a EGFR.CAR followed by a P2Asequence, which was followed by a sequence encoding CD16 (orCD16^(158V)), and which in turn was followed by an IRES sequence elementupstream of a sequence encoding erIL-2. Unless otherwise noted, theEGFR.CAR had a structure as exemplarily shown in FIG. 1 (however, withan Fc epsilon signaling sequence) and had a nucleic acid sequence of SEQID NO:33. Of course, it should be noted that other suitable sequencesfor the production of EGFR.CAR may have a sequence identity of at least90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%,or at least 99% to SEQ ID NO:33. The primary transcript of thetricistronic nucleic acid then lead to the formation of EGFR.CAR,CD16^(158V), and erIL-2 as the recombinant polypeptides. FIG. 16exemplarily and schematically depicts a linearized recombinant nucleicacid used for transfection of the NK-92 cells.

The EGFR.CAR and CD16 expression on the NK-92 cell surface wasdetermined by flow cytometry using biotinylated anti-scFv antibody andAPC-labeled streptavidin, and fluorescently labeled anti-CD16 antibody.Exemplary results for EGFR.CAR and CD16 expression (polyclonal) areshown in FIG. 17 along with aNK (not expressing CD16) and haNK(expressing CD16) controls. As can be readily seen from FIG. 17, thepolyclonal cell cultures had a significant and strong expression forboth, EGFR.CAR and CD16^(158V). In this context, it should once more benoted that the EGFR.CAR was expressed at notably higher levels where theHER2.CAR had a FcεRIγ signaling portion as compared to other signalingportions (data not shown).

Natural cytotoxicity was determined for polyclonal cell culture of theEGFR.CAR t-haNK cells, and FIG. 18 depicts an exemplary set of results.Here, the natural cytotoxicity against K562 cells indeed exceeded thecytotoxicity of aNK control cells. Similarly, CAR-mediated cytotoxicitywas determined for polyclonal cell culture of the EGFR.CAR t-haNK cellsagainst HCT116 target cells and against A549 cells, both of whichexpressed EGFR. As can be seen from the graphs in FIG. 19, CAR-mediatedcytotoxicity was significant and specific. In still further experiments,the inventors tested EGFR.CAR t-haNK polyclonal cells for ADCC againstSUP-B15^(EGFR−/CD20+) cells using rituximab as target specific antibodyand Herceptin as control antibody. As can be taken from FIG. 20, allEGFR.CAR t-haNK cells had once more significant, strong, and targetspecific ADCC versus control.

After dilution propagation, selected EGFR.CAR t-haNK cell clones weretested for natural cytotoxicity against 562 cells. Notably, all selectedEGFR.CAR t-haNK cell clones had substantially identical naturalcytotoxicity which was comparable to the aNK control as can be takenfrom FIG. 21. Selected EGFR.CAR t-haNK cell clones were then tested forCAR-mediated cytotoxicity against HCT116 tumor cells, and exemplaryresults are shown in the graphs of FIG. 22. Once more all EGFR.CARt-haNK cell clones had very significant CAR-mediated cytotoxicityagainst HCT116 tumor cells approaching 90% specific lysis. Similarresults were obtained using A549 tumor cells as target cells as is shownin FIG. 23.

In still further experiments, the inventors determined ADCC for selectedEGFR.CAR t-haNK cell clones using SUP-B15^(EGFR−/CD20+) cells as targetcells, rituximab as target specific antibody, and Herceptin as controlantibody. As already observed with the polyclonal cell culture and ascan be seen from FIG. 24, selected EGFR.CAR t-haNK cell clones exhibitedeffective ADCC in the presence of cognate antibodies.

While all EGFR.CAR t-haNK polyclonal cultures and cell clones propagatedwell in the absence of exogenous IL-2, expression of er-IL2 was alsotested along with the quantity of erIL-2 released in the culture medium.Exemplary results are shown in FIG. 25 depicting for some clones a minorrelease of erIL-2 into the culture medium while other clones had anerIL-2 release comparable to control. As noted above, reduced erIL-2release may further advantageously reduce potential systemic effectsotherwise attributable to IL-2 (e.g., vascular permeability, systemicvascular leak, etc.).

Example 3: Quadracistronic Constructs

While the above examples were performed with tricistronic constructs itshould be appreciated that the same constructs can also be implementedin a quadracistronic construct to express IL-12, a TGF-beta trap, or ahoming receptor to so reduce immune suppression in the tumormicroenvironment and enrich the tumor microenvironment with thuslymodified NK cells. One exemplary quadracistronic construct isschematically illustrated in FIG. 26 where a nucleic acid sequenceencoding a TGF-beta trap is upstream of the tricistronic construct asdiscussed above. As can be seen from FIG. 26, a P2A sequence is locatedbetween the TGF-beta trap and the CAR to ensure coordinated expressionwhile producing distinct proteins. Expression of the TGF-beta trap wasthen tested in an ELISA to ascertain that the quadracistronic constructindeed produced a functional TGF-beta trap, and FIG. 27 depictsexemplary results versus control. Notably, expression levels of theTGF-beta trap were significant throughout all recombinant clones withquadracistronic construct.

Similarly, quadracistronic constructs were prepared using a secretedIL-12 single chain heterodimer as is schematically depicted in FIG. 28.Once more, a P2A sequence was placed between the IL-12 single chainheterodimer sequence and the CAR sequence. FIG. 29 shows exemplaryembodiments of the single chain heterodimer with alternate order of p35and p40 subunits (which were both separated by a linker sequence). Ascan be seen from the expression levels tested, both single chainheterodimers expressed equally well in the cells tested relative tocontrol. In a still further embodiment, a quadracistronic construct wasprepared using CCR7 as a homing receptor and an exemplaryquadricictronic arrangement is depicted in FIG. 30.

Of course, it should be recognized that for all nucleic acid sequencesprovided herein the corresponding encoded proteins are also expresslycontemplated herein. Likewise, for all amino acid sequences,corresponding nucleic acids sequences are also contemplated herein (withany codon usage).

All patent applications, publications, references, and sequenceaccession numbers cited in the present specification are herebyincorporated by reference in their entirety.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

In this specification and in the claims that follow, reference will bemade to a number of terms that shall be defined to have the followingmeanings:

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

It is understood that all numerical values described herein (e.g., pH,temperature, time, concentration, amounts, and molecular weight,including ranges) include normal variation in measurements encounteredby one of ordinary skill in the art. Thus, numerical values describedherein include variation of +/−0.1 to 10%, for example, +/−0.1%, 0.5%,1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. It is to be understood,although not always explicitly stated, that all numerical designationsmay be preceded by the term “about.” Thus, the term about includesvariation of +/−0.1 to 10%, for example, +/−0.1%, 0.5%, 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, or 10% of the numerical value. It is also to beunderstood, although not always explicitly stated, that the reagentsdescribed herein are merely exemplary and that equivalents of such areknown in the art.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein include the end points of the range, and includeall values between the end points of the range. All ranges disclosedherein also encompass any and all possible subranges and combinations ofsubranges thereof. Any listed range can be easily recognized assufficiently describing and enabling the same range being broken downinto at least equal halves, thirds, quarters, fifths, tenths, etc. As anon-limiting example, each range discussed herein can be readily brokendown into a lower third, middle third and upper third, etc. As will alsobe understood by one skilled in the art all language such as “up to,”“at least,” and the like, include the number recited and refer to rangeswhich can be subsequently broken down into subranges as discussed above.Finally, as will be understood by one skilled in the art, a rangeincludes each individual member. Thus, for example, a group having 1-3cells refers to groups having 1, 2, or 3 cells. Similarly, a grouphaving 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and soforth.

It is also to be understood, although not always explicitly stated, thatthe reagents described herein are merely exemplary and that equivalentsof such are known in the art.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not.

The term “comprising” is intended to mean that the compositions andmethods include the recited elements, but not excluding others.“Consisting essentially of,” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination. For example, a composition consistingessentially of the elements as defined herein would not exclude otherelements that do not materially affect the basic and novelcharacteristic(s) of the claimed invention. “Consisting of” shall meanexcluding more than trace amount of other ingredients and substantialmethod steps recited. Embodiments defined by each of these transitionterms are within the scope of this disclosure.

As used herein, “immunotherapy” refers to the use of NK-92 cells,modified or unmodified, naturally occurring or modified NK cell orT-cell, whether alone or in combination, and which are capable ofinducing cytotoxicity when contacting a target cell.

As used herein, “natural killer (NK) cells” are cells of the immunesystem that kill target cells in the absence of a specific antigenicstimulus, and without restriction according to major histocompatibilitycomplex (MHC) class. Target cells may be tumor cells or cells harboringa virus. NK cells are characterized by the presence of CD56 and theabsence of CD3ζ surface markers.

The term “endogenous NK cells” is used to refer to NK cells derived froma donor (or the patient), as distinguished from the NK-92 cell line.Endogenous NK cells are generally heterogeneous populations of cellswithin which NK cells have been enriched. Endogenous NK cells may beintended for autologous or allogeneic treatment of a patient.

The term “NK-92” refers to natural killer cells derived from the highlypotent unique cell line described in Gong et al. (1994), rights to whichare owned by NantKwest (hereafter, “NK-92™ cells”). The immortal NK cellline was originally obtained from a patient having non-Hodgkin'slymphoma. Unless indicated otherwise, the term “NK-92™” is intended torefer to the original NK-92 cell lines as well as NK-92 cell lines thathave been modified (e.g., by introduction of exogenous genes). NK-92™cells and exemplary and non-limiting modifications thereof are describedin U.S. Pat. Nos. 7,618,817; 8,034,332; 8,313,943; 9,181,322; 9,150,636;and published U.S. application Ser. No. 10/008,955, all of which areincorporated herein by reference in their entireties, and include wildtype NK-92™ NK-92™-CD16, NK-92™-CD16-γ, NK-92™-CD16-ζ,NK-92™-CD16(F176V), NK-92™MI, and NK-92™CI. NK-92 cells are known topersons of ordinary skill in the art, to whom such cells are readilyavailable from NantKwest, Inc.

The term “aNK” refers to an unmodified natural killer cells derived fromthe highly potent unique cell line described in Gong et al. (1994),rights to which are owned by NantKwest (hereafter, “aNK™ cells”). Theterm “haNK” refers to natural killer cells derived from the highlypotent unique cell line described in Gong et al. (1994), rights to whichare owned by NantKwest, modified to express CD16 on the cell surface(hereafter, “CD16+NK-92™ cells” or “haNK® cells”). In some embodiments,the CD16+NK-92™ cells comprise a high affinity CD16 receptor on the cellsurface. The term “taNK” refers to natural killer cells derived from thehighly potent unique cell line described in Gong et al. (1994), rightsto which are owned by NantKwest, modified to express a chimeric antigenreceptor (hereafter, “CAR-modified NK-92™ cells” or “taNK® cells”). Theterm “t-haNK” refers to natural killer cells derived from the highlypotent unique cell line described in Gong et al. (1994), rights to whichare owned by NantkWest, modified to express CD 16 on the cell surfaceand to express a chimeric antigen receptor (hereafter, “CAR-modifiedCD16+ NK-92™ cells” or “t-haNK™ cells”). In some embodiments, thet-haNK™ cells express a high affinity CD16 receptor on the cell surface.

A “modified NK-92 cell” refers to an NK-92 cell that expresses anexogenous gene or protein, such as an Fc receptor, a CAR, a cytokine(such as IL-2 or IL-15), and/or a suicide gene. In some embodiments, themodified NK-92 cell comprises a vector that encodes for a transgene,such as an Fc receptor, a CAR, a cytokine (such as IL-2 or IL-15),and/or a suicide gene. In one embodiment, the modified NK-92 cellexpresses at least one transgenic protein.

As used herein, “non-irradiated NK-92 cells” are NK-92 cells that havenot been irradiated. Irradiation renders the cells incapable of growthand proliferation. It is envisioned that the NK-92 cells will beirradiated at the treatment facility or some other point prior totreatment of a patient, since the time between irradiation and infusionshould be no longer than four hours in order to preserve optimalactivity. Alternatively, NK-92 cells may be prevented from proliferatingby another mechanism.

As used herein, “inactivation” of the NK-92 cells renders them incapableof growth. Inactivation may also relate to the death of the NK-92 cells.It is envisioned that the NK-92 cells may be inactivated after they haveeffectively purged an ex vivo sample of cells related to a pathology ina therapeutic application, or after they have resided within the body ofa mammal a sufficient period of time to effectively kill many or alltarget cells residing within the body. Inactivation may be induced, byway of non-limiting example, by administering an inactivating agent towhich the NK-92 cells are sensitive.

As used herein, the terms “cytotoxic” and “cytolytic,” when used todescribe the activity of effector cells such as NK-92 cells, areintended to be synonymous. In general, cytotoxic activity relates tokilling of target cells by any of a variety of biological, biochemical,or biophysical mechanisms. Cytolysis refers more specifically toactivity in which the effector lyses the plasma membrane of the targetcell, thereby destroying its physical integrity. This results in thekilling of the target cell. Without wishing to be bound by theory, it isbelieved that the cytotoxic effect of NK-92 cells is due to cytolysis.

The term “kill” with respect to a cell/cell population is directed toinclude any type of manipulation that will lead to the death of thatcell/cell population.

The term “Fc receptor” refers to a protein found on the surface ofcertain cells (e.g., natural killer cells) that contribute to theprotective functions of the immune cells by binding to part of anantibody known as the Fc region. Binding of the Fc region of an antibodyto the Fc receptor (FcR) of a cell stimulates phagocytic or cytotoxicactivity of a cell via antibody-mediated phagocytosis orantibody-dependent cell-mediated cytotoxicity (ADCC). FcRs areclassified based on the type of antibody they recognize. For example,Fc-gamma receptors (FCγR) bind to the IgG class of antibodies. FCγRIII-Ais a low affinity Fc receptor bind to IgG antibodies and activate ADCC.FCγRIII-A are typically found on NK cells. NK-92 cells do not expressFCγRIII-A. Fc-epsilon receptors (FcεR) bind to the Fc region of IgEantibodies.

The term “chimeric antigen receptor” (CAR), as used herein, refers to anextracellular antigen-binding domain that is fused to an intracellularsignaling domain. CARs can be expressed in T cells or NK cells toincrease cytotoxicity. In general, the extracellular antigen-bindingdomain is a scFv that is specific for an antigen found on a cell ofinterest. A CAR-expressing NK-92 cell is targeted to cells expressingcertain antigens on the cell surface, based on the specificity of thescFv domain. The scFv domain can be engineered to recognize any antigen,including tumor-specific antigens and virus-specific antigens. Forexample, CD19CAR recognizes CD19, a cell surface marker expressed bysome cancers.

The term “tumor-specific antigen” as used herein refers to antigens thatare present on a cancer or neoplastic cell but not detectable on anormal cell derived from the same tissue or lineage as the cancer cell.Tumor-specific antigens, as used herein, also refers to tumor-associatedantigens, that is, antigens that are expressed at a higher level on acancer cell as compared to a normal cell derived from the same tissue orlineage as the cancer cell.

The term “virus-specific antigen” as used herein refers to antigens thatare present on a virus-infected cell but not detectable on a normal cellderived from the same tissue or lineage as the virus-infected cell. Inone embodiment, a virus-specific antigen is a viral protein expressed onthe surface of an infected cell.

The terms “polynucleotide”, “nucleic acid” and “oligonucleotide” areused interchangeably and refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides or analogsthereof. Polynucleotides can have any three-dimensional structure andmay perform any function, known or unknown. The following arenon-limiting examples of polynucleotides: a gene or gene fragment (forexample, a probe, primer, EST or SAGE tag), exons, introns, messengerRNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probesand primers. A polynucleotide can comprise modified nucleotides, such asmethylated nucleotides and nucleotide analogs. If present, modificationsto the nucleotide structure can be imparted before or after assembly ofthe polynucleotide. The sequence of nucleotides can be interrupted bynon-nucleotide components. A polynucleotide can be further modifiedafter polymerization, such as by conjugation with a labeling component.The term also refers to both double- and single-stranded molecules.Unless otherwise specified or required, any embodiment of this inventionthat is a polynucleotide encompasses both the double-stranded form andeach of two complementary single-stranded forms known or predicted tomake up the double-stranded form.

A polynucleotide is composed of a specific sequence of four nucleotidebases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil(U) for thymine when the polynucleotide is RNA. Thus, the term“polynucleotide sequence” is the alphabetical representation of apolynucleotide molecule.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology canbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare homologous at that position. A degree of homology between sequencesis a function of the number of matching or homologous positions sharedby the sequences.

As used herein, “percent identity” refers to sequence identity betweentwo peptides or between two nucleic acid molecules. Percent identity canbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare identical at that position. Homologous nucleotide sequences includethose sequences coding for naturally occurring allelic variants andmutations of the nucleotide sequences set forth herein. Homologousnucleotide sequences include nucleotide sequences encoding for a proteinof a mammalian species other than humans. Homologous amino acidsequences include those amino acid sequences which contain conservativeamino acid substitutions and which polypeptides have the same bindingand/or activity. In some embodiments, a homologous amino acid sequencehas no more than 15, nor more than 10, nor more than 5 or no more than 3conservative amino acid substitutions. In some embodiments, a nucleotideor amino acid sequence has at least 60%, at least 65%, at least 70%, atleast 80%, or at least 85% or greater percent identity to a sequencedescribed herein. In some embodiments, a nucleotide or amino acidsequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identity to a sequence described herein. Percent identity can bedetermined by, for example, the Gap program (Wisconsin Sequence AnalysisPackage, Version 8 for UNIX, Genetics Computer Group, UniversityResearch Park, Madison Wis.), using default settings, which uses thealgorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).Algorithms suitable for determining percent sequence identity includethe BLAST and BLAST 2.0 algorithms, which are described in Altschul etal. (Nuc. Acids Res. 25:3389-402, 1977), and Altschul et al. (J. Mol.Biol. 215:403-10, 1990), respectively. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (see the internet at ncbi.nlm.nih.gov). TheBLAST algorithm parameters W, T, and X determine the sensitivity andspeed of the alignment. The BLASTN program (for nucleotide sequences)uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5,N=−4 and a comparison of both strands. For amino acid sequences, theBLASTP program uses as defaults a wordlength of 3, and expectation (E)of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc.Natl. Acad. Sci. USA 89:10915, 1989) alignments (B) of 50, expectation(E) of 10, M=5, N=−4.

In some embodiments, a nucleic acid sequence is codon optimized forexpression in a particular species, for example, a mouse sequence can becodon optimized for expression in humans (expression of the proteinencoded by the codon-optimized nucleic acid sequence). Thus, in someembodiments, a codon-optimized nucleic acid sequence has at least 60%,at least 65%, at least 70%, at least 80%, or at least 85% or greaterpercent identity to a nucleic acid sequence described herein. In someembodiments, a codon-optimized nucleic acid sequence acid sequence hasat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity toa sequence described herein.

The term “express” refers to the production of a gene product (e.g., aprotein). The term “transient” when referring to expression means apolynucleotide is not incorporated into the genome of the cell. The term“stable” when referring to expression means a polynucleotide isincorporated into the genome of the cell, or a positive selection marker(i.e., an exogenous gene expressed by the cell that confers a benefitunder certain growth conditions) is utilized to maintain expression ofthe transgene.

The term “cytokine” or “cytokines” refers to the general class ofbiological molecules which affect cells of the immune system. Exemplarycytokines include but are not limited to interferons and interleukins(IL)—in particular IL-2, IL-12, IL-15, IL-18 and IL-21. In preferredembodiments, the cytokine is IL-2.

As used herein, the term “vector” refers to a non-chromosomal nucleicacid comprising an intact replicon such that the vector may bereplicated when placed within a permissive cell, for example by aprocess of transformation. A vector may replicate in one cell type, suchas bacteria, but have limited or no ability to replicate in anothercell, such as mammalian cells. Vectors may be viral or non-viral.Exemplary non-viral vectors for delivering nucleic acid include nakedDNA; DNA complexed with cationic lipids, alone or in combination withcationic polymers; anionic and cationic liposomes; DNA-protein complexesand particles comprising DNA condensed with cationic polymers such asheterogeneous polylysine, defined-length oligopeptides, and polyethyleneimine, in some cases contained in liposomes; and the use of ternarycomplexes comprising a virus and polylysine-DNA. In one embodiment, thevector is a viral vector, e.g. adenovirus. Viral vectors are well knownin the art.

As used herein, the term “targeted,” when referring to proteinexpression, is intended to include, but is not limited to, directingproteins or polypeptides to appropriate destinations in the cell oroutside of it. The targeting is typically achieved through signalpeptides or targeting peptides, which are a stretch of amino acidresidues in a polypeptide chain. These signal peptides can be locatedanywhere within a polypeptide sequence, but are often located on theN-terminus. Polypeptides can also be engineered to have a signal peptideon the C-terminus. Signal peptides can direct a polypeptide forextracellular section, location to plasma membrane, golgi, endosomes,endoplasmic reticulum, and other cellular compartments. For example,polypeptides with a particular amino acid sequence on their C-terminus(e.g., KDEL) are retained in the ER lumen or transported back the ERlumen.

As used herein, the term “target,” when referring to targeting of atumor, refers to the ability of NK-92 cells to recognize and kill atumor cell (i.e., target cell). The term “targeted” in this contextrefers, for example, to the ability of a CAR expressed by the NK-92 cellto recognize and bind to a cell surface antigen expressed by the tumor.

As used herein, the term “transfect” refers to the insertion of nucleicacid into a cell. Transfection may be performed using any means thatallows the nucleic acid to enter the cell. DNA and/or mRNA may betransfected into a cell. Preferably, a transfected cell expresses thegene product (i.e., protein) encoded by the nucleic acid.

The term “suicide gene” refers to a transgene that allows for thenegative selection of cells expressing that transgene. A suicide gene isused as a safety system, allowing the cells expressing the gene to bekilled by introduction of a selective agent. A number of suicide genesystems have been identified, including the herpes simplex virusthymidine kinase (TK) gene, the cytosine deaminase gene, thevaricella-zoster virus thymidine kinase gene, the nitroreductase gene,the Escherichia coli gpt gene, and the E. coli Deo gene (see also, forexample, Yazawa K, Fisher W E, Brunicardi F C: Current progress insuicide gene therapy for cancer. World J. Surg. 2002 July; 26(7):783-9).In one embodiment, the suicide gene is the thymidine kinase (TK) gene.The TK gene may be a wild-type or mutant TK gene (e.g., tk30, tk75,sr39tk). Cells expressing the TK protein can be killed usingganciclovir.

What is claimed is:
 1. A genetically modified NK cell transfected withone or more recombinant nucleic acids encoding and expressing (i) amembrane bound recombinant chimeric antigen receptor (CAR) thatcomprises in a single polypeptide chain an extracellular binding domainhaving SEQ ID NO:41, a hinge domain, a transmembrane domain, and aFcεRIγ signaling domain having the polypeptide sequence of SEQ ID NO:1,wherein the extracellular binding domain specifically binds to EGFR;(ii) a recombinant CD16; (iii) an autocrine growth stimulating cytokine.2. The genetically modified NK cell of claim 1 wherein the NK cell is anNK-92 cell.
 3. The genetically modified NK cell of claim 1 wherein therecombinant CD16 is a CD16^(158V) mutant.
 4. The genetically modified NKcell of claim 1 wherein the autocrine growth stimulating cytokine isIL-2 or IL-15.
 5. The genetically modified NK cell of claim 4 whereinthe autocrine IL-2 or IL-15 further comprises an endoplasmic retentionsequence.
 6. The genetically modified NK cell of claim 1 wherein the oneor more recombinant nucleic acids further encodes and the cell expressesone of IL-12, a TGF-beta trap, or a homing receptor.
 7. The geneticallymodified NK cell of claim 6 wherein the IL-12 is a single chain IL-12heterodimer.
 8. The genetically modified NK cell of claim 6 wherein theTGF-beta trap comprises a single chain dimer of the TGF-beta Receptor IIectodomain.
 9. The genetically modified NK cell of claim 8 wherein theTGF-beta trap is a secreted form of a single chain dimer of the TGF-betaReceptor II ectodomain.
 10. The genetically modified NK cell of claim 6wherein the homing receptor is a cell adhesion molecule, a selectin, anintegrin, a C-C chemokine receptor, or C-X-C chemokine receptor.
 11. Thegenetically modified NK cell of claim 10 wherein the homing receptor isselected from the group consisting of CCR1, CCR2, CCR3, CCR4, CCR5,CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6,CXCR7, CX3 CR1, XCR1, CCXCKR, D6, DARC, and a receptor for CXCL14.
 12. Agenetically modified NK cell, comprising: a recombinant nucleic acidencoding (i) a membrane bound recombinant chimeric antigen receptor(CAR) that comprises in a single polypeptide chain an extracellularbinding domain having SEQ ID NO:41, a hinge domain, a transmembranedomain, and a FcεRIγ signaling domain having the polypeptide sequence ofSEQ ID NO:1, wherein the extracellular binding domain specifically bindsto EGFR; (ii) a recombinant CD16; (iii) an autocrine growth stimulatingcytokine.
 13. The genetically modified NK cell of claim 12 wherein therecombinant nucleic acid is a polycistronic DNA.
 14. The geneticallymodified NK cell of claim 12 wherein the NK cell is an NK-92 cell. 15.The genetically modified NK cell of claim 12 wherein the recombinantCD16 is a CD16^(158V) mutant.
 16. The genetically modified NK cell ofclaim 12 wherein the autocrine growth stimulating cytokine is IL-2 orIL-15.
 17. The genetically modified NK cell of claim 16 wherein theautocrine IL-2 or IL-15 further comprises an endoplasmic retentionsequence.
 18. The genetically modified NK cell of claim 12 wherein therecombinant nucleic further encodes one of IL-12, a TGF-beta trap, or ahoming receptor.
 19. The genetically modified NK cell of claim 18wherein the IL-12 is a single chain IL-12 heterodimer.
 20. Thegenetically modified NK cell of claim 18 wherein the TGF-beta trapcomprises a single chain dimer of the TGF-beta Receptor II ectodomain.21. The genetically modified NK cell of claim 20 wherein the TGF-betatrap is a secreted form of a single chain dimer of the TGF-beta ReceptorII ectodomain.
 22. The genetically modified NK cell of claim 18 whereinthe homing receptor is a cell adhesion molecule, a selectin, anintegrin, a C-C chemokine receptor, or C-X-C chemokine receptor.
 23. Thegenetically modified NK cell of claim 22 wherein the homing receptor isselected from the group consisting of CCR1, CCR2, CCR3, CCR4, CCR5,CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCRS, CXCR6,CXCR7, CX3 CR1, XCR1, CCXCKR, D6, DARC, and a receptor for CXCL14.
 24. Amethod of treating cancer in a patient in need thereof, comprisingadministering to the patient a therapeutically effective amount of thegenetically modified NK cells of claim 1 thereby treating the cancer.25. The method of claim 24 further comprising a step of administering atleast one additional therapeutic entity selected from the groupconsisting of a viral cancer vaccine, a bacterial cancer vaccine, ayeast cancer vaccine, N-803, an antibody, a stem cell transplant, and atumor targeted cytokine.
 26. The method of claim 24, wherein the canceris a lung cancer, a breast cancer, a thyroid cancer, an esophagealcancer, a gastric cancer, a gastroesophageal cancer, or a head and neckcancer.
 27. The method of claim 24, wherein about 1×10⁸ to about 1×10¹¹cells per m² of body surface area of the patient are administered to thepatient.